A solid-phase synthesis method which uses the phosphoramidite method is broadly used in the chemical synthesis of oligonucleotide. In this method, for example, a nucleoside which becomes the 3′-terminal of the oligonucleotide to be synthesized is firstly loaded in advance on a solid-phase support via a cleavable linker such as succinyl group, and this support is put into a reaction column and set on an automatic oligonucleotide synthesizer. Thereafter, synthesizing reagents are fed into the reaction column, for example, in the following manner in accordance with the synthesizing program of the automatic oligonucleotide synthesizer. (1) Deprotection of nucleoside 5′-OH group by a trichloroacetic acid/dichloromethane solution, dichloroacetic acid/toluene solution or the like, (2) coupling reaction of amidite with the 5′-OH group by a nucleoside phosphoramidite (nucleoside monomer)/acetonitrile solution and an activator (tetrazole or the like)/acetonitrile solution, (3) capping of the unreacted 5′-OH group by acetic anhydride/pyridine/methyl imidazole/THF or the like, and (4) oxidation of phosphite by iodine/water/pyridine or the like.
By repeating this synthesis cycle, oligonucleotide having the intended sequence is synthesized. The finally synthesized oligonucleotide is cut out from the solid-phase synthesis support by hydrolyzing the cleavable linker with ammonia, methylamine or the like (cf. non-patent document 1).
As the solid-phase support to be used in the oligonucleotide synthesis, inorganic particles such as CPG (Controlled Pore Glass), silica gel and the like have so far been used, but in recent years, resin beads which can increase quantity of oligonucleotide synthesized per weight of solid-phase support have been started to be used for synthesis at a moderate price and in a large quantity. As such resin beads, a highly-crosslinked and non-swelling porous polystyrene bead (cf. patent document 1), a low-crosslinked and swelling porous polystyrene bead (cf. patent document 2) and the like can for example be mentioned.
Generally, a longer oligonucleotide chain synthesized problematically causes lower synthesizability (synthetic purity and synthetic quantity). To solve this, it is necessary to decrease the loading amount of a nucleoside linker to be the origin of the synthesis of a solid-phase support. For example, DNA oligonucleotide containing 20 bases can be synthesized with high purity by using a commercially available porous resin bead solid-phase support having a nucleoside linker loading amount of about 200 μmol/g. However, in the case of a DNA oligonucleotide containing 40 bases, the amount needs to be 80 μmol/g or less. Also, when an RNA oligonucleotide or a modified oligonucleotide is to be synthesized, since amidite containing a bulky protecting group or modifying group is coupled, the oligonucleotide is synthesized using a lower loading amount of a nucleoside linker to prevent the synthesizability from decreasing.
However, when an oligonucleotide is synthesized using a porous resin bead as a solid-phase support for oligonucleotide synthesis, and a decreased laoding amount of a nucleoside linker, unexpectedly, the synthesizability problematically becomes lower than expected.